Caspase-6 (Casp6) is implicated in Alzheimer disease (AD) cognitive impairment and pathology. Hippocampal atrophy is associated with cognitive impairment in AD. Here, a rare functional exonic missense CASP6 single nucleotide polymorphism (SNP), causing the substitution of asparagine with threonine at amino acid 73 in Casp6 (Casp6N73T), was associated with hippocampal subfield CA1 volume preservation. Compared to wild type Casp6 (Casp6WT), recombinant Casp6N73T altered Casp6 proteolysis of natural substrates Lamin A/C and α-Tubulin, but did not alter cleavage of the Ac-VEID-AFC Casp6 peptide substrate. Casp6N73T-transfected HEK293T cells showed elevated Casp6 mRNA levels similar to Casp6WT-transfected cells, but, in contrast to Casp6WT, did not accumulate active Casp6 subunits nor show increased Casp6 enzymatic activity. Electrophysiological and morphological assessments showed that Casp6N73T recombinant protein caused less neurofunctional damage and neurodegeneration in hippocampal CA1 pyramidal neurons than Casp6WT. Lastly, CASP6 mRNA levels were increased in several AD brain regions confirming the implication of Casp6 in AD. These studies suggest that the rare Casp6N73T variant may protect against hippocampal atrophy due to its altered catalysis of natural protein substrates and intracellular instability thus leading to less Casp6-mediated damage to neuronal structure and function.
Caspase-6 (Casp6), a cysteine protease that cleaves its protein substrates after an aspartic acid residue, is associated with Alzheimer disease (AD) pathogenesis. Active Casp6, Casp6-cleaved Tau (Tau∆Casp6), Casp6-cleaved α-Tubulin (Tub∆Casp6), and Casp6-cleaved valosin containing protein p97 (p97∆Casp6) are detected in neurofibrillary tangles (NFT), neuritic plaques and neuropil threads of sporadic and familial AD and mild cognitively impaired (MCI) brains, but absent in young control brains1,2,3,4. Active Casp6 and Tau∆Casp6 are observed in pre- and mature NFT, but only Tau∆Casp6 is still present in ghost tangles5,6. Surprisingly, in aged non-cognitively impaired (NCI) brains, Casp6 activity is detected uniquely in the inter-neuronally connected entorhinal cortex (ERC) and hippocampal Cornu Ammonis 1 (CA1)1,5,7, the first areas of the brain presenting with NFT according to Braak staging8,9. Furthermore, active Casp6 is detected in AD pathologies of the anterior olfactory bulb, whose neurons project their axons into the ERC10. Therefore, Casp6 activity is an early spatial–temporal event in AD pathogenesis.
Casp6 activity is associated with age-dependent cognitive impairment. In aged NCI individuals, high levels of Casp6 correlate with global cognitive scores and predict lower episodic and semantic memory performance, the first types of memories impaired in AD1,7,11. Casp6 activity in the anterior olfactory bulb is associated with lower global cognitive scores, mini mental state exam (MMSE) scores, and episodic and semantic memories10. Cerebrospinal fluid Tau∆Casp6 levels reflect human brain Tau∆Casp6 levels, and correlate with AD severity, and global cognitive, MMSE, episodic, semantic and working memory scores12. Furthermore, expression of self-activated human Casp6 in mouse CA1 neurons is sufficient to induce age-dependent episodic and spatial memory impairment11.
Active Casp6 is associated with selective neuronal degeneration, but not necessarily cell death. Casp6 activation in HEK293T cells does not induce cell death13,14. Microinjection of recombinant active Casp6 in human neurons in primary cultures induces a dose-dependent and protracted type of cell death15. Similarly, overexpression of wild type or mutant forms of amyloid precursor protein (APP) associated with familial AD induces Casp6-dependent, but amyloid beta peptide (Aβ) independent, neurodegeneration in human neurons16. This eventually leads to neuronal cell death that is Aβ dependent. Nerve growth factor (NGF)-deprived mouse primary dorsal root ganglia and sympathetic neuron cultures leads to compartmentalized Casp6-dependent axonal degeneration and Caspase-3 (Casp3)-dependent cell death17,18,19. In AD brains, active Casp6-immunopositive neurons do not show apoptotic features and active Casp3 immunoreactivity is sparse6,20,21. Furthermore, transgenic expression of a self-activated human Casp6 in mouse brains causes hippocampal CA1 neurodegeneration, defined here as the loss of neuronal structure or function, in the absence of significant neuronal loss11,22. Consistently, microinjected active Casp6 causes neuronal degeneration and impairs synaptic transmission in CA1 pyramidal neurons, but not in medium spiny neurons22.
Casp6 cleaves many protein substrates such as α-Tubulin, Tau, α-Actinin-4, Drebrin, Glial fibrillary acidic protein, Spinophilin, and Vimentin which are critical for neuronal structure and function and synaptic plasticity2,5,23,24. Casp6-cleaved valosin containing protein p97 impairs ubiquitin proteasome system-mediated protein degradation3. Ubiquitin ligases of mouse double minute 2 homolog, ubiquitin fusion degradation 2, and developmentally down-regulated protein 4, proteins involved in cellular protein turnover are also Casp6 substrates25,26. Furthermore, Casp6 cleaves APP, Presenilin 1 and 2, and Huntingtin, proteins involved in neurodegenerative diseases27,28,29. These neuronal substrates reflect Casp6 potential function in neurodegeneration.
Casp6 is activated by overexpression and proteolytic processing. Human Casp6 is expressed as an inactive zymogen from the CASP6 gene30 and the level of Casp6 protein is relatively low in normal human brain31. Prokaryotic and eukaryotic overexpression of Casp6 is sufficient for self-proteolytic activation14,32,33. CASP6 transcriptional regulation has not been extensively investigated, but p53 is a CASP6 transcription regulator in neurons and astrocytes34,35,36. p53 levels are enhanced in familial AD neurons37, indicating a mechanism for increased CASP6 gene expression. The Casp6 active site consists of catalytic cysteine 163 (C163) surrounded by four binding pockets for substrate selectivity38. Casp6 maturation requires three cleavage events at D23, D179, and D193 to remove the pro-domain and the inter-subunit linker between a large subunit (LS) and a small subunit (SS), so a small β-sheet structure 190TEVD193 in the inter-subunit linker occupying the active site is removed and catalytic C163 is exposed33. Proteolytic processing allows the correct conformation of exosites and allosteric sites important for the regulation of catalytic specificity and efficiency39,40. Casp6 can be processed and activated by itself at high concentration14,32, by Casp3 in apoptotic cells41, or by Caspase-1 in stressed primary human neurons42,43. Interestingly, p53 also transcriptionally regulates CASP1 gene expression44, which can also self-activate, indicating possible co-regulated expression of CASP1 and CASP6 genes.
Many genetic risk factors have been identified for AD, but no genetic association between CASP6 and AD has been reported. Here, we used SKAT-O analysis45 to identify whether rare functional exonic CASP6 variants associate with human brain volumes, determined by MRI scans in aged individuals. A rare variant, Casp6N73T, associated with hippocampal subfield CA1 volumes in ADNI. Recombinant Casp6N73T exhibited altered proteolysis of natural protein substrates compared to wild type Casp6 (Casp6WT), limited enzymatic activity and stability in cell culture, and eliminated Casp6-mediated dysfunction and degeneration in hippocampal CA1 pyramidal neurons. These results suggest that Casp6N73T may provide a protective effect against hippocampal atrophy compared to Casp6WT.
Genetic association between the Caspase-6 gene (CASP6) and hippocampal CA1 volume in AD
From the Alzheimer’s Disease Neuroimaging Initiative (ADNI) whole genome sequencing (WGS) data (n = 757), only one functional exonic missense SNP (rs141550898) with a minor allele frequency < 0.05 was identified within CASP6. We performed SKAT-O of rs141550898 with CA1 volumes with age, sex, year of education, MRI field strength, and intracranial volume as covariates. One individual (male, early MCI, APOE ε3/ε3) was heterozygous for this variant. The optimized sequence kernel association test (SKAT-O) yielded a significant association of rs141550898 in CASP6 with hippocampal CA1 subfield volume (p = 0.0146). The rs141550898 SNP in which AAT is replaced by ACT causes a substitution of asparagine (N) for threonine (T) at amino acid 73 of Casp6, thus generating Casp6N73T. The participant carrying Casp6N73T has maintained its early MCI status for at least 5 year based on his last visit, raising the possibility that Casp6N73T may slow down hippocampal CA1 atrophy and AD progression.
Gene expression of CASP6 and CASP1
To confirm the implication of CASP1 and CASP6 in the brains of individuals assessed in the ADNI cohort, CASP1 and CASP6 mRNA levels were evaluated. The number, age, sex, mean age at death, and apolipoprotein APO E genotype of the participants used for differential gene expression analysis in each brain region are summarized in Table 1. The RNA integrity number (RIN) where 10 represents intact RNA and 1 completely degraded RNA ranged from 6.3 to 8.7. Gene expression association analysis using RNA-Seq data generated from temporal cortex (TCX), para-hippocampal gyrus (PHG), inferior frontal gyrus (IFG), superior temporal gyrus (STG), frontal pole (FP), dorsolateral prefrontal cortex (DLPFC), and cerebellum (CER) identified that CASP6 mRNA was up-regulated in the TCX (Fig. 1a), STG (Fig. 1b), PHG (Fig. 1c), and DLPFC (Fig. 1d) of AD compared to cognitively normal older adults. Levels in the CER (Fig. 1e), FP (Fig. 1f), and IFG (Fig. 1g) were not significantly different. Casp6 is activated by Caspase-1 (Casp1), therefore CASP1 mRNA levels were also evaluated. CASP1 mRNA levels were significantly up-regulated in AD TCX (Fig. 1h) and STG (Fig. 1i), but not in PHG (Fig. 1j), DLPFC (Fig. 1k), CER (Fig. 1l), FP (Fig. 1m), or IFG (Fig. 1n), compared to cognitively normal older adults.
Recombinant Caspase-6 N73T (Casp6N73T) zymogen showed similar self-processing as Caspase-6 wild type (Casp6WT) zymogen
To observe the self-processing of Caspase-6 N73T (Casp6N73T) zymogen, recombinant protein from three independent clones expressing Casp6N73T zymogen were compared with Caspase-6 wild type (Casp6WT) zymogen and catalytically inactive triple mutant Casp6D(23,179,193)A. The Casp6WT zymogen undergoes self-processing at D23, D179, and D193 to remove the pro-domain and the inter-subunit linker, which allows the formation of active Casp6 consisting of two large subunits (LS) and two small subunits (SS) (Fig. 2a). Purified recombinant Casp6N73T and Casp6WT zymogens both generated the expected small subunit (SS) and large subunit with (LSL) or without (LS) inter-subunit linker. Casp6WT and Casp6N73T zymogens generated more LSL than LS (Fig. 2b) indicating preferred self-cleavage at D193, as previously observed with Casp6WT14,33,46. The catalytically inactive Casp6D(23,179,193)A was not processed, as expected, and migrated as full-length (FL) Casp6 at 34 kDa. The Casp6N73T zymogen generated LSL, LS, and SS at levels comparable to those of the Casp6WT zymogen (Fig. 2b,c). FL, LSL, LS, and SS of Casp6 were confirmed with western blotting against anti-FL and anti-LSL Casp6 antiserum (Fig. 2d,e), neoepitope antiserum recognizing Casp6 cleaved at D179 (Fig. 2f,g), and anti-SS antibodies (Fig. 2h,i). No difference in the levels of each subunit between Casp6WT and Casp6N73T was detected (Fig. 2e,g,i). These results indicate that Casp6N73T zymogen self-processing is similar to that of Casp6WT zymogen.
Recombinant Casp6N73T showed comparable VEIDase activity as Casp6WT on the tetrapeptide Ac-VEID-AFC substrate
To determine whether Casp6N73T affects Casp6 catalysis, the catalytic efficiency of Casp6N73T on fluorescent tetrapeptide Ac-VEID-AFC was compared to Casp6WT. Before the activity assay, the number of active sites was established for each enzyme preparation by active site titration against de-esterified irreversible pan-caspase inhibitor, zVAD-FMK, to ensure comparison of equal amounts of Casp6WT and Casp6N73T active sites (Supplementary Fig. S1a-f). Two to four hundred nM active Casp6N73T exhibited comparable VEIDase activity (defined as cleaved pmol AFC per minute) than similar concentrations of active Casp6WT (Fig. 3a). The velocities of product formation by 2, 10, and 50 nM Casp6N73T with 20 µM Ac-VEID-AFC substrate were similar to those of 2, 10, or 50 nM Casp6WT (Fig. 3b). Furthermore, to better understand the kinetics of Casp6N73T, the Km, Vmax, kcat and kcat/Km were determined based on Michaelis–Menten kinetics measurements (Supplementary Fig. S2). No differences were found in the Km, Vmax, kcat or kcat/Km between Casp6N73T and Casp6WT (Fig. 3c).
Recombinant Casp6N73T showed increased proteolytic processing of natural Casp6 protein substrate, Lamin A/C
To assess if Casp6N73T affects Casp6 activity on natural protein substrates, the cleavage of Lamin A/C by Casp6N73T and Casp6WT was assessed. Lamin A/C was extracted from Casp6 KO mouse tissue, and was incubated with varying concentrations of active site titrated Casp6N73T or Casp6WT. Both Casp6N73T and Casp6WT processed Lamin A/C and generated the expected 28 kDa fragment (Fig. 4a)47. The % FL Lamin A/C and cleaved Lamin A/C generated by Casp6N73T at concentrations from 50 to 500 nM were similar to that of Casp6 WT after 1 h incubation (Fig. 4b). In order to calculate the initial reaction velocity, cleaved Lamin A/C was measured every 5 min for the first 15 min of reaction, followed by every 15 min until 60 min. The amount of cleaved Lamin A/C increased with incubation time with both Casp6N73T and Casp6WT (Fig. 4c). Initially, at the 5 and 10 min time points, 200–500 nM Casp6N73T consistently generated slightly higher amounts of cleaved Lamin A/C than Casp6WT (Fig. 4d). The slope of the linear phase of the curve in Fig. 4d determined the initial velocity of the enzymes on Lamin A/C. The initial velocity of Casp6N73T was significantly higher than that of Casp6WT (Fig. 4e). These results suggest that the N73T substitution may modify Casp6N73T interaction with Lamin A/C.
Recombinant Casp6N73T showed decreased catalytic efficiency on α-Tubulin
To further understand the effect of Casp6N73T on different protein substrates, the catalytic activity of Casp6N73T and Casp6WT on α-Tubulin was compared. Both Casp6N73T and Casp6WT processed α-Tubulin and generated a fragment 2 kDa smaller than the FL (Fig. 5a top panel), consistent with Casp6 cleavage at VGVD438 in α -Tubulin. Cleavage of α-tubulin at D438 was confirmed with the neoepitope antibody GN20622 (Fig. 5a bottom panel). The % Tub∆Casp6 by Casp6N73T at concentration 15.6–250 nM was significantly lower than that of Casp6WT after 4 h incubation (Fig. 5b). Kinetically, 15.6–62.5 nM Casp6N73T consistently generated less Tub∆Casp6 compared to Casp6WT during the first 4 h of incubation (Fig. 5c&d). Analysis of the initial velocity determined by the slope of the linear phase of the cleavage curve in Fig. 5d, showed a significant 50% reduction of Casp6N73T catalytic efficiency (Fig. 5e). These results show that Casp6N73T catalytic efficiency on α-Tubulin is lower than that of Casp6WT. As observed with the Lamin A/C substrate, the results suggest that Casp6N73T may interact differently than Casp6WT with α-Tubulin.
The steady state level of active Casp6N73T subunits is considerably lower than those of Casp6WT in transfected human embryonic kidney 293 T (HEK293T) cells
To assess if eukaryotically expressed Casp6N73T behaves like the prokaryotically expressed recombinant Casp6N73T, CASP6WT and CASP6N73T cDNAs lacking their pro-domain (∆Pro) to promote self-processing of Casp6 were cloned in pCep4β and the constructs were transfected in human embryonic kidney 293 T (HEK293T) cells. Catalytically inactive, CASP6C163A with its pro-domain sequence, was transfected as a control for full length Casp6. Casp6 mRNA levels were equivalent in CASP6WT, CASP6N73T, or CASP6C163A cDNA transfected cells (Fig. 6a,b). In transfected cells, the ∆ProCasp6 expressed in CASP6WT- and CASP6N73T-transfected cells migrated approximately 2 kDa below the full length Casp6 in CASP6C163A-transfected cells (Fig. 6c). Unexpectedly, CASP6N73T-transfected HEK293T cells contained consistently lower levels of ∆Pro-Casp6 than CASP6WT-transfected cells (Fig. 6c,d). Compared to CASP6C163A-transfected cells, ∆ProCasp6 levels were lower in CASP6WT-transfected cells, as expected since Casp6WT can be processed into its subunits (Fig. 6d). The levels of LS in CASP6N73T-transfected cells were significantly lower than those of CASP6WT-transfected cells (Fig. 6e,f). Consistently, CASP6N73T-transfected HEK293T protein extracts did not show significant Casp6 VEIDase activity compared to non-, mock-, or empty vector-transfected cell extracts, while CASP6WT-transfected cells exhibited high VEIDase activity (Fig. 6g). Because transfected cells contained similar Casp6WT and Casp6N73T mRNA levels, decreased Casp6N73T full length protein level and VEIDase activity may be the result by enhanced turnover. Indeed, in the presence of translational inhibitor cycloheximide (CHD), the degradation rate of ∆ProCasp6N73T was faster than that of ∆ProCasp6WT levels (Fig. 6h,i), suggesting a higher cellular turnover of Casp6 due to the N73T substitution. Since Casp6 LS has previously been reported to be degraded by the proteasome13,48, we verified if proteasomal activity may be responsible for FL or LS Casp6N73T lower levels. The epoxomicin proteasomal inhibitor did not significantly alter levels of either Casp6N73T FL or LS in HEK293T cells, excluding proteasomal degradation as an explanation (Supplementary Fig. S3). Nevertheless, these results indicate that Casp6N73T is unstable and degraded by an alternate non-proteasomal dependent mechanism, thereby limiting the amount of Casp6N73T LS and Casp6N73T activity produced in mammalian cells.
Recombinant Casp6N73T caused less neurofunctional damage and neuronal degeneration than Casp6WT in hippocampal CA1 pyramidal neurons
The effect of Casp6N73T on neuronal function was compared with recombinant Casp6WT and catalytically inactive Casp6C163A proteins by patching CA1 pyramidal neurons in hippocampal organ slices with these proteins (Fig. 7a) and measuring the amplitude of excitatory postsynaptic potential (EPSP) (Fig. 7b). EPSP amplitudes from recorded neurons were analyzed when the membrane potential and input resistance were stable (Fig. 7b bottom panel). Activated Casp6WT and Casp6N73T were stable for 5 h after their preparation in internal solution (Supplementary Fig. S4), within which the recording data was acquired. Ten pg Casp6WT induced a decrease of EPSP amplitude 30 min after patching, and a continually decreasing EPSP amplitude during the 50 min of recording (Fig. 7c). In contrast 10 pg catalytically inactive Casp6C163A did not alter EPSP amplitude within 50 min. Interestingly, 10 pg Casp6N73T decreased the EPSP amplitude less than Casp6WT in pyramidal neurons for approximately 10 min after patching, and EPSP amplitude was maintained at 65% of the original levels for 50 min (Fig. 7c). Paired pulse ratio (∆PPR) of the first and second EPSP wave increased in Casp6WT patched neurons compared to Casp6C163A, suggesting that the decaying EPSP amplitude was at least in part due to a reduced probability of release presynaptically49.
In contrast, Casp6N73T ∆PPR was indistinguishable from that of Casp6C163A (Fig. 7d), possibly hinting at an absence of perceivable effects on presynaptic release. Although the change in PPR due to Casp6WT suggested a presynaptic locus of action, the coefficient of variation (CV) analysis of the first EPSP50 indicated a predominantly postsynaptic effect, although with variable outcome (Fig. 7e). Taken together, the combined outcome of PPR and CV analyses is consistent with a coordinated downregulation on both pre- and post-synaptic sides due to Casp6WT.
The morphology of Casp6WT, Casp6N73T, or Casp6C163A patched CA1 pyramidal neurons was analyzed by manual 3D reconstruction (Fig. 7f). Dendritic map density and convex hulls indicated the average distribution and the maximum extent of dendrites, respectively (Fig. 7g). Neurons patched with Casp6WT exhibited a lower length of apical dendrites in the SLM region compared to Casp6C163A-patched neurons, but the data did not reach statistical significance (Fig. 7h). The apical dendrites length of Casp6N73T-patched neurons was longer than those of Casp6WT-patched neurons. In addition, Casp6WT-patched neurons displayed beading along basal neurites in the stratum oriens region indicative of neuronal degeneration (Fig. 7i), while beading was rarely seen in neurons patched with Casp6C163A, indicating that this beading is linked to Casp6 activity. The beading dendrites of Casp6N73T-patched neurons was approximately 50% lower than in neurons patched with Casp6WT. These results indicate that Casp6N73T is less detrimental to neuron function and causes less neurodegeneration than Casp6WT.
Our study demonstrates that a rare human CASP6 variant encoding Casp6N73T, genetically associated with preserved hippocampal CA1 volume in an AD cohort, exhibits altered catalyses on natural protein substrates, lamin A and α-Tubulin. In addition, our study showed that Casp6N73T has less negative impact on neuronal function and neurodegeneration than the Casp6WT in mice CA1 hippocampal pyramidal neurons. These results provide initial evidence for a neuroprotective effect by Casp6N73T as reflected by hippocampal subfield volume preservation but this needs to be validated in independent data sets.
The significant association between Casp6N73T and hippocampal CA1 volume by SKAT-O provides an advantage in identifying associations between low frequency functional exonic SNPs and pathological features of AD compared to genome-wide association studies (GWAS)45,51. Different from the single regression model used in GWASs, SKAT uses a multiple regression model to regress the phenotypes of different SNPs in the same genomic region and allows different orientations of the phenotype, in order to assess the cumulative effects of one gene on disease. Another advantage of our approach is the use of a quantitative measure of hippocampal CA1 volume, instead of the numerous variables of clinically diagnosed AD. Hippocampal CA1 volume was chosen because atrophy in this region occurs early in AD progression52,53. Large scale longitudinal measures indicate that hippocampal CA1 atrophy is a robust MRI biomarker to distinguish MCI that further develop to AD from stable MCI54. Furthermore, rare SNPs also mean lower number of individuals carrying the SNP, therefore, our finding will require testing additional cases to confirm the role of Casp6N73T in MCI and AD. On the other hand, the ADNI study provides precise cognitive and neuroimaging information on the individual carrying CASP6N73T. The Casp6N73T variant was identified in a heterozygous individual at the early MCI stage with unexpected preserved hippocampal CA1 volume compared to those carrying the wild type CASP6.
Here, Casp6N73T was shown to be less damaging to synaptic transmission and plasticity than Casp6WT. In contrast to catalytically inactive Casp6C163A, Casp6WT rapidly decreased induced EPSP amplitude in CA1 hippocampal neurons, whereas Casp6N73T showed only a minor decrease. In addition, the paired pulse ratio was increased in Casp6WT, but not in Casp6N73T. Furthermore, Casp6WT, but not Casp6C163A, induced basal dendritic beading indicating Casp6 activity-mediated neurodegeneration CA1 neurons, a feature that was reduced with Casp6N73T. Based on the morphological analysis of reconstructed neurons, Casp6WT, but not Casp6N73T, caused a decrease in the total apical dendritic length. These data are consistent with transgenic expression of a self-activated form of Casp6WT in CA1 neurons causing age-dependent cognitive impairment11, small amounts of active Casp6 causing a rapid depression of CA1 neuronal transmission22 and impairing long-term potentiation in hippocampal CA1 circuits in vivo55. Given that the presence of active Casp6 detected in the entorhinal cortex of aged individuals correlates with decreased cognitive performance1,7,12,42 and that early cognitive decline is strongly correlated with synaptic loss56,57, these data raise the possibility that the active Casp6 observed in neuropil threads, neuritic plaques and neurofibrillary tangles4,5 causes synaptic transmission problems. Most importantly, the data suggest that Casp6N73T variant would not be as detrimental as Casp6WT for neuronal structure and function and this may protect aged individuals from cognitive decline. In support of this conclusion, Casp6 deficient mice have been shown to be protected against excitotoxicity, nerve growth factor deprivation and myelin-induced axonal degeneration, and showed an age-dependent increase in cortical and striatal volume58.
The molecular reason for the protective action of Casp6N73T might stem from its altered proteolysis of natural protein substrates compared to Casp6WT. Casp6 can cleave numerous protein substrates, some of which are specific to neurons and synapses2,59 and involved in neurodegenerative diseases, such as Tau, Vimentin, Drebrin, Spinophilin, α-Actinin2,5, valosin containing protein p973, APP60, Presenilin 1 and 228, and Huntingtin61, that may be responsible for structural damage, synaptic loss, and protein aggregation. Here, we show that compared to Casp6WT, Casp6N73T had increased proteolytic processing of Lamin A/C, decreased proteolysis of α-tubulin and equivalent processing of the Ac-VEID-AFC peptide substrate. These results indicate that either the N73T alters the structure of the Casp6 enzyme thereby affecting the catalytic site, or the N73T alters the interaction of Casp6 with substrates. The altered catalysis of natural protein substrates α-Tubulin and Lamin A/C, but not of the small peptide substrate, suggests that the active site of the Casp6N73T is relatively normal. Therefore, it is likely that Casp6N73T interacts differently with α-Tubulin and Lamin A/C than Casp6WT, thus suggesting the presence of an exosite either within N73, its surroundings, or in a structure that is altered by N73T. Exosites are defined as sites outside the substrate-binding catalytic site which influence structure or function of an enzyme. The N73T substitution is located in the middle of Casp6’s helix B with a shorter side chain pointing outward. The nearby negatively charged D72 is conserved among Casp6 orthologues and unique to Casp6 among the caspase protein family, which may also contribute to this exosite. Casp6 exosite 42RRR44, located at the hinge between the core structure and the N-terminus of the large subunit, has been confirmed62,63. Furthermore, two rare human variants, G66R and R65W completely eliminate or significantly reduce Casp6 activity through impaired substrate binding, alter the catalytic site activity, and have dominant negative effects on Casp6 WT46. The alternatively spliced Casp6b isoform lacking amino acids 13–104 while retaining the catalytic site also has no activity, and inhibits Casp6WT activation64. The N73T data presented here further highlights the importance of the N-terminus of the large subunit of Casp6 in substrate recognition. Casp6N73T may change the proteolysis of different neuronal substrates in addition to α-Tubulin, which together display a reduced damage due to Casp6 and favors a neuroprotective phenotype. Future investigation in characterizing the cleavage efficiencies of Casp6N73T on these substrates could expand our understanding of the new exosite as well as the protective mechanism of Casp6N73T. The identification of an additional exosite for Casp6 could be useful in specific drug design against Casp6 activity, which has been shown to cause age-dependent cognitive impairment in mice11.
The protective action of Casp6N73T might also stem from its instability in mammalian cells. Casp6N73T full length levels were decreased significantly relative to Casp6WT, despite similar mRNA levels for Casp6N73T and Casp6WT. This lower level of full length Casp6N73T could be explained by either a lower mRNA translational rate, increased protein processing, or increased degradation by alternate cellular proteolytic activities. A more rapid turnover for full length Casp6N73T compared to FL Casp6WT was observed indicating either increased protein processing into its active subunits or increased degradation by alternate cellular proteolytic activities. The fact that the level of Casp6N73T processed large subunit (LS) was reduced almost tenfold relative to Casp6WT indicated that Casp6N73T was processed less efficiently than Casp6WT or that the Casp6N73T LS was degraded more rapidly than the Casp6WT LS. Proteasomal activity was excluded since proteasomal inhibition did not significantly alter either FL or LS Casp6N73T levels. The fact that self-processing of prokaryotically expressed recombinant Casp6N73T is equivalent to that of Casp6WT eliminates the possibility that the Casp6N73T mutation alters processing. Therefore, the most likely explanation for these findings is that the FL Casp6N73T is unstable and degraded by a non-proteasomal cellular mechanism, thereby limiting the amount of Casp6N73T LS and Casp6N73T activity produced in mammalian cells.
Lastly, we confirm here that CASP1 and CASP6 mRNA levels are significantly increased in specific regions of the AD brains in the ADNI cohort compared to cognitively normal control brains from large sample sizes in the AMP-AD Consortium. We investigated CASP1 expression because it activates Casp6 in human primary neurons42. Increased CASP1 mRNA levels have been reported previously in AD cortex and entorhinal cortex (ERC)65,66. Our results additionally show increased CASP1 mRNA levels in AD temporal cortex and superior temporal gyrus. CASP6 expression in AD is more controversial. One early study reports low level of CASP6 mRNAs in AD brains65. Others indicate increased CASP6 mRNA levels in cortex and cerebellum of AD brains43,66. Here, we show increased CASP6 mRNA levels in AD temporal cortex, superior temporal gyrus, para-hippocampal gyrus, and dorsolateral prefrontal cortex. These results support the implication of CASP6 in AD.
This study highlights the importance of assessing the role of the amazing genetic diversity of humans in disease by combining human genetic information associated with well-ascertained neuroimaging and cognitive measures with biochemical and electrophysiological approaches to investigate the potential influence of rare variants on AD-related pathologies and cognition.
Alzheimer’s Disease Neuroimaging Initiative (ADNI)
Individuals used in the analysis were ADNI participants o67,68. Inclusion and exclusion criteria, clinical and neuroimaging protocols, and other information about ADNI can be found at www.adni-info.org and http://www.loni.usc.edu/ADNI/. Written informed consent was obtained at the time of enrollment for imaging and genetic sample collection and protocols of consent forms were approved by each participating sites’ Institutional Review Board. Human subject ethical approval was obtained by ADNI and can be found at http://www.loni.usc.edu/ADNI/. All methods and experiments were performed in accordance with relevant guidelines.
Whole genome sequencing (WGS) analysis
WGS on the Illumina HiSeq2000 platform with paired-end reads was performed on blood-derived genomic DNA samples obtained from 817 ADNI participants69. Briefly, short-read sequences were mapped to the human genome assembly (GRCh build 37.72) using BWA70. During the alignment, we use only bases with Phred Quality > 15 in each read to include soft clipping of low-quality bases, retain only uniquely mapped pair-end reads, and remove potential PCR duplicates. After completing initial alignment, the alignment was further refined by locally realigning any suspicious reads. The reported base calling quality scores obtained from the sequencer were re-calibrated to account for covariates of base errors. All variants with statistical evidence for an alternate allele present among individuals were identified using GATK HaplotypeCaller for multi-sample variant callings.
Baseline T1-weighted brain MRI scans were downloaded from the ADNI database. FreeSurfer software was used to process T1-weighted brain MRI scans71 and extract region of interest (ROI)-based imaging phenotypes72,73.
Gene-based association analysis
Since population stratification is known to cause spurious association in disease studies, we restricted our analyses to only subjects that clustered with CEU (Utah residents with Northern and Western European ancestry from the CEPH collection) + TSI (Tuscany in Italy) populations using HapMap 3 genotype data and the multidimensional scaling analysis (www.hapmap.org)74,75,76. A total of 757 ADNI participants (259 CN, 219 early MCI, 232 late MCI, and 47 AD) were used for analysis, where late and early MCI were defined as the cognitive performance below 1.5 standard deviations of the normative mean on a standard test and at the range of 1 to 1.5 standard deviations, respectively77. After extracting WGS-identified functional exonic SNPs within CASP6, we performed a gene-based association analysis of rare variants (minor allele frequency < 0.05) using SKAT-O software78. For hippocampal CA1 volumes, age, sex, year of education, MRI field strength, and total intracranial volume were used as covariates.
RNA-Seq data (n = 1,966 individuals for the seven brain regions) reprocessed and realigned in the AMP-AD Consortium using a RNA-Seq pipeline79, were downloaded from the Sage Bionetworks (www.synapse.org). Human postmortem brain RNA-Seq data were obtained in three independent studies (Religious Orders Study and the Memory and Aging Project (ROS/MAP), Mount Sinai School of Medicine (MSSM), and Mayo Clinic) from seven distinct brain regions. Differential gene expression of CASP6 and CASP1 between AD and cognitively normal controls were analysed with R package limma80.
Mutagenesis and sub-cloning of Casp6N73T
The pET23b( +)-Casp6WT-His plasmid (Addgene #11,823 https://www.addgene.org/11823/, gift from Dr. Guy Salvesen, Burnham Institute, La Jolla, CA, USA) encodes a human Casp6WT zymogen fused with a C-terminal 6 × His tag. The mammalian pCEP4β- Casp6∆ProWT-His plasmid encodes a human Casp6WT lacking its pro-domain14. Both plasmids were mutated using QuikChange Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA, USA) with Casp6N73T primers 5'- GCG CAG ATA GAG ACA CTC TTA CCC GCA GG -3’ and 5'- CCT GCG GGT AAG AGT GTC TCT ATC TGC GC -3' to introduce the mutation for the N73T substitution. All constructs were verified by sequencing at McGill University Genome Quebec Innovation Center.
Protein expression and purification
The pET23b( +)-Casp6WT-His or pET23b( +)-Casp6N73T-His plasmids were expressed in BL21(DE3)pLysS competent cells (Promega, Madison, WI, USA). Casp6 was purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Chicago, IL, USA) and Macro Prep High Q Resin (Bio-Rad Laboratories, Hercules, CA, USA) as described32,46. Pure protein was aliquoted, fast frozen in EtOH/dry ice bath, and stored at − 80 °C freezer.
Active site titration
Casp6 active site concentration was determined using an irreversible inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone (zVAD-FMK; MP Biomedicals, Irvine, CA, USA) as described46.
Caspase activity assays on Ac-VEID-AFC
The release of AFC from 20 µM Ac-VEID-AFC by active site titrated Casp6 (2, 10, 20, 50, 100, or 400 nM) was measured as described46. The slope of the linear phase of cleaved AFC plotted against time was calculated as VEIDase activity.
Determination of K m and k cat
Release of AFC from 1–200 µM Ac-VEID-AFC by 20 nM Casp6 at 37 °C in Stennicke buffer was measured as described46. VEIDase activity versus Ac-VEID-AFC concentration was fitted to a Michaelis–Menten equation v = (Vmax × [S])/(Km + [S]) using Prism 7 software (GraphPad Software, CA, USA) and maximal velocity Vmax, Michaelis–Menten constant Km , and kcat = Vmax/[active site concentration of Casp6] were calculated.
Casp6 activity assay on protein substrates
To assess Lamin A/C cleavage, nuclear proteins were extracted from Casp6 knockout mice colon tissue81 as described46. Casp6 (50–500 nM) were incubated with 3 µg lysate in Stennicke buffer at 37 °C for 5–60 min.
To assess α-Tubulin cleavage, purified porcine Tubulin (Cytoskeleton, Inc., Denver, CO, USA) was used. Tubulin powder was reconstituted in General Tubulin Buffer (80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5 mM EGTA) supplemented with 10 mg/mL GTP. Casp6 (15.6–250 nM) was incubated with 252 nM Tubulin in Stennicke buffer at 37 °C for 1–12 h.
After the incubation period, samples were prepared in Laemmli buffer and analyzed by Western blot. The initial velocity of Casp6 on Lamin A/C or α-Tubulin was calculated based on the linear portion of the cleavage curve. The number of data points in the linear portion of each cleavage curve used for linear regression depended on the goodness-of-fit when R2 was closest to 1.00.
Fifty ng recombinant Casp6, 6 µg nuclear or 20 µg HEK293T protein extracts, 0.05–0.2 µg Tubulin, were submitted to western blot analyses with 1:1000 mouse anti-Casp6 against human Casp6 amino acids 24–293 (SC-81653, Santa Cruz Biotechnology, Dallas, TX, USA), 1:10,000 rabbit neoepitope 10630 antiserum5, or 1:250 mouse anti-Casp6 small subunit (BD Pharmingen clone B93-4, BD Biosciences, San Jose, CA, USA), 1:1000 anti-Lamin A/C (Cell Signaling Technology #2032, Danvers, MA, USA), 1;1000 anti-cleaved Lamin A/C at VEID230 (Cell Signaling Technology #2035), 1;1000 anti- α-Tubulin (Cell Signaling Technology #11H10), 1:5000 anti-cleaved α-Tubulin at VGVD43814, or 1:5000 anti-β-Actin ( Sigma-Aldrich Co # A5441, Oakville, ON, Canada). Immunoreactivity was detected with 1:5000 HRP-conjugated anti-rabbit (DAKO P0217, Agilent Technologies Burlington, ON, Canada) or anti-mouse (Jackson ImmunoResearch Labs #133499, West Grove, PA, USA) secondary antibody followed by ECL (GE Healthcare), or with alkaline phosphatase-conjugated anti-mouse secondary antibody (1:5000; Jackson) followed by NBT/BCIP (Promega). Immunoreactive bands were scanned, and densitometry was performed using ImageJ software (NIH, Bethesda, MD, USA) by measuring band intensity values beyond the background.
Transfection, treatments, and protein extraction of HEK293T cells
The transfections were carried out with Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada) and proteins extracted after 24 h in cell lysis buffer for Casp6 activity or in RIPA for western blots as described14. Transfection efficiency of Lipofectamine 2000 was optimized to obtain more than 90% transfected cells by calculating the number of fluorescent cells transfected with pBud-EGFP plasmid (Addgene #23027) over the number of total cells stained with Hoechst (Thermo Fisher Scientific). For protein degradation analysis, cells were treated 24 h after transfection with 75 µg/ml cycloheximide (CHD, Sigma-Aldrich) for the indicated times.
Treatment of transfected HEK293T cells with epoxomicin
Cells were treated with 50 nM of epoxomicin (Enzo) 24 h after transfection for the indicated times and proteins extracted in cell lysis buffer. Proteasomal inhibition was confirmed in a proteasome activity assay as reported previously48 using 50 µM Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC; Enzo).
Total RNA was extracted using TRIzol (Invitrogen) and converted to cDNA with avian myeloblastosis reverse transcriptase (Roche Diagnostics, Laval, QC, Canada). Casp6 cDNA was amplified with Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA) as described46.
All animal experimentation was approved by the McGill University Animal Care Committee and performed under guidelines and regulations in accordance with the ARRIVE guidelines. Hippocampal slices (300 µm) from 11–16 days old C57BL/6 mice were prepared in ice-cold ACSF22.
Recombinant Casp6 were activated in Stennicke buffer at 37 °C for 15 min, and diluted to 10 pg/10 µL in internal solution82 with 30–60 µM Alexa 594 or 40–80 µM Alexa 488 (Invitrogen), filtered through 0.22 µm hydrophilic polyethersulfone filters (Millipore Sigma, Oakville, ON, Canada), and osmolarity double checked to be ~ 310 mOsm.
Multiple whole-cell patches were performed as described82. The stimulating electrode was placed in the CA1 stratum orients and delivered five 0.1-µs-long biphasic pulses (10–50 V) at 30 Hz every 30 s to elicit EPSPs in the range 1–3 mV. The recording was done for 1 h unless the data failed to meet the stability criteria defined as potential (± 4 mV), input resistance (± 15%) and temperature (32–34 °C). Offline data were analysed with Igor Pro (WaveMetrics Inc.). EPSP peak amplitudes were measured and averaged every 2.5 min (5 traces). Ensemble time courses were normalized to the first 2.5 min EPSP.
Two-photon imaging and neuron reconstruction
Two-photon excitation was achieved using a Chameleon Ultra II femtosecond laser (Coherent, Santa Clara, CA, USA) tuned to 780 nm for both Alexa 594 and 488. Two-photon microscopes were custom-built83. Imaging data were acquired using customized versions of ScanImage 2018 (Vidrio Technologies, Leesburg VA USA)84 running in Matlab (The MathWorks, Natick, MA, USA) via a PCI-6110 or a PCIe-6374 data acquisition board (National Instruments, Austin, TX, USA). When neurons had been well loaded with dye (> 1 h after break-in), the neuronal morphology was acquired as stacks of 512-by-512-pixels slices at 2 ms/line spaced by 2 µm. Maximum-intensity two-photon-imaging stacks compiled with ImageJ (NIH) were used for morphological identification (Fig. 7a), and for quantification of basal dendrite beading. Imaging montage of entire neurons was performed by Affinity Designer 1.7 (Serif Ltd, West Bridgford, Notts, UK). Image stacks were used for manual reconstruction of 3D morphologies (Fig. 7f) using the Neuromantic freeware(http://www.reading.ac.uk/ neuromantic/body_index.php). Morphometry of 3D reconstructions (e.g. density maps, hulls, etc. in Fig. 7g,h) was subsequently performed using custom software83 running in Igor Pro 8 (WaveMetrics Inc.). CA1 layer boundaries were identified using laser-scanning Dodt contrast images acquired simultaneously with 2-photon fluorescence.
Statistical analyses of data were performed using Igor Pro 8 or GraphPad Prism 7 with Student’s t-test or one/two-way ANOVA, as indicated in figure legends.
Materials generated for this study are available upon request. The datasets supporting the conclusions of this article are available in ADNI database (http://www.loni.usc.edu/ADNI/) for whole genome sequencing and MRI scan data the Sage Bionetworks (www.synapse.org) for human postmortem brain RNA-Seq data from seven brain regions.
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This work was supported by funds from the Canadian Institutes of Health Research (CIHR) 2011MOP-243413-BCA-CGAG-45097 and 201610PJT-377052-PJT-CFAF-45097, Leaders Opportunity Fund, Canadian Foundation for Innovation and Jewish General Hospital Foundation to ALB. LZ is a recipient of an Alzheimer Society of Canada Dr. and Mrs. Spatz doctoral award. PJS was funded by CFI LOF 28331, CIHR OG 126137, FRSQ CB 254033, and NSERC DG/DAS 2017-04730. NC was in receipt of NSERC USRA 552184-2020 and FRQNT BPCA Supplement 298265. MH was funded by Canada Summer Jobs 2020. KN was funded by NLM R01 LM012535 and NIA R03 AG054936, and AJS by NIA R01 AG19771, NIA P30 AG10133, NLM R01 LM011360, DOD W81XWH-14-2-0151, and NIGMS P50GM115318. We would like to thank Dr. Guy Klaiman for the preparation of recombinant Casp6D23,179,193A, Dr. Bénédicte Foveau for the dissection of colon tissue from Casp6 knockout mice, and Dr. Hovy Ho-Wai Wang, Airi Watanabe, and Christina Chou from PJS lab for maintaining the mice used for electrophysiology. Data collection and sharing for this project was funded by the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (National Institutes of Health Grant U01AG024904) and DODADNI (Department of Defense award number W81XWH-12-2-0012). ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the following: AbbVie, Alzheimer’s Association; Alzheimer’s Drug Discovery Foundation; Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Cogstate; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Roche Ltd and its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research & Development LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer Inc.; Piramal Imaging; Servier; Takeda Pharmaceutical Company; and Transition Therapeutics. The Canadian Institutes of Health Research is providing funds to support ADNI clinical sites in Canada. Private sector contributions are facilitated by the Foundation for the National Institutes of Health (http://www.fnih.org). The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimer’s Therapeutic Research Institute at the University of Southern California. ADNI data are disseminated by the Laboratory for Neuro Imaging at the University of Southern California. The ROSMAP study data were provided by the Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago. Data collection was supported through funding by National Institute on Aging (NIA) grants P30AG10161, R01AG15819, R01AG17917, R01AG30146, R01AG36836, U01AG32984, and U01AG46152, the Illinois Department of Public Health, and the Translational Genomics Research Institute. The Mayo RNA-seq study data were provided by the following sources: the Mayo Clinic Alzheimer’s Disease Genetic Studies, led by Dr. Nilufer Ertekin-Taner and Dr. Steven G. Younkin, Mayo Clinic, Jacksonville, Florida, using samples from the Mayo Clinic Study of Aging, the Mayo Clinic Alzheimer’s Disease Research Center, and the Mayo Clinic Brain Bank. Data collection was supported through funding by NIA grants P50 AG016574, R01 AG032990, U01 AG046139, R01 AG018023, U01 AG006576, U01 AG006786, R01 AG025711, R01 AG017216, and R01 AG003949; National Institute of Neurological Disorders and Stroke (NINDS) grant R01 NS080820; the CurePSP Foundation; and support from Mayo Foundation. Study data include samples collected through the Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona. The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026, National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the NIA (P30 AG19610, Arizona Alzheimer’s Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901, and 1001 to the Arizona Parkinson’s Disease Consortium), and the Michael J. Fox Foundation for Parkinson’s Research. The Mount Sinai Brain Bank (MSBB) data were generated from postmortem brain tissue collected through the Mount Sinai VA Medical Center Brain Bank and were provided by Dr. Eric Schadt of the Mount Sinai School of Medicine through funding from NIA grant U01AG046170. Data used in preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database (http://adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A complete listing of ADNI investigators can be found at: http://adni.loni.usc.edu/wp-content/uploads/how_to_apply/ADNI_Acknowledgement_List.pdf.
The authors declare no competing interests.
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Zhou, L., Nho, K., Haddad, M.G. et al. Rare CASP6N73T variant associated with hippocampal volume exhibits decreased proteolytic activity, synaptic transmission defect, and neurodegeneration. Sci Rep 11, 12695 (2021). https://doi.org/10.1038/s41598-021-91367-0